Ring a aromatized steroidal compounds and process for preparing them



Patented Sept. 2, 1952 RING A AROMATIZED STEROIDAL COM- POUNDS AND PROCESS FOR PREPAR mo; THEM Maximilian R. Ehrenstein, Philadelphia, Pa., as-

signor to The Trustees "of The University'ofv Pennsylvania, Philadelphia,Pa., a corporation of Pennsylvania ,No Drawing. AppIicationMa'y ZG,1950,

Serial No. 164,634 2 Claims. (01. 260-3971) The present invention relates to the prep-aration :of ring A aromatized steroidal compounds, and more particularly to the preparation of esitratr'iene, compounds, which have a physiological activity similar to the 'estrogenic hormones. The present invention involves anovel method of preparing compounds of this type, and the new compounds obtained by this process.

It is. therefore, a general object of. the present invention to provide an improved process for the preparation of estra-triene compounds, and

a specific object to provide a process whereby the aromatization .of ring A of a steroidal compound can be efiected under mild conditions. A further object is to provide new compounds having estrog enlike activity, Broadly, the invention involves the preparation of compounds having the general formula CH3 000R f wherein R is an esterifying-radical such as an alkyl, aralkyl or aryl radical; and R1 is hydrogen or an acyl radical, and particularly those compounds wherein R is a lower alkyl radical, such as the methyl, ethyl, propyl, butyl, amyl and the like radicals; and R1 is derived from a low molecular weight fatty acid, such as acetic acid,- by either treating 3'(;3),5,19-trihydroxy etiochloanate with Rany nickel under mild conditions or by subjecting 3-(13) ,l9-diacetoxy-5-hydroxyetiocholanic acid to high-vacuum distill-ation and hydrolyzing the estatriene thus formed to a 3-hydroxy-estratriene-l'I-carboxylate.

From reports of other workers in the art, it

was e'xpected that on distilling in a high vacuum, 3(5),19-diacyl-5-hydroxyetiocholanic acid would undergo dehydration leading to 3(),19-diacy l- M-etiocholenic acid; This assumption was fnrther supported by the fact that 3'(fi)-=acyloxy- A -etiocholenic acid can be distilled in a highvacuum without undergoing any decomposition. It was surprisingly discovered, however, that when distilled under high vacuum, 3(;3),19-diacy1oxy-5-hydroxyetiocholanic acid yielded 3- acyloxy-A -:es=tratriene-17-oarboxylic acid.- Similarly, in counter-distinction to recent repoi' tsvdescri'bing the catalytic conversion of pregnenolone to progesterone-and the oxidation of additional steroids, such as cholesterol, dihy-drocholesterol and gepicoprostan-ol by the use of a specialRaney nickel and cyclohexanone, it was surprisingly found .that' when 3(B),5,19,-trihydroxyf'ztiochol-anates were treated with ordinary Rianey nickel and a hydrogen acceptor such as =cyclohexanone, the entire group attached to vthe lo position c'arbonatom wasr-emoved, andthe A ring was completely aromatized. The result of this vreaction was all the more surprising since any primary hydroxyl group at'carbon atom 19 was expected .to be converted to an aldehyde group, which latter group would. immediately condense with the oyclohexanone, as has been described in a number of. instances of the, 011- penauer reaction. While the mechanism of the aromatization i s not known with certainty, it is reasonable, to-assume that dehydrogenation 0c;- cur first at carbon atom 3 yielding the 3-keto derivative of 5,19-dihydroxy etiocholanate, which might then have been dehydrated to the 3-keto- 19-hydroxy A- etiocholenate. The mechanism of the loss of carbqnatom 19 is not clear. It is possible tha-t one molecule of methanol is split off directly, or it is possible that first: a dehy:

occurs; The intermediate 3 eketo .-A fi estradiene-l'l-carboxylate, a dienone, will rearrange 3 to the 3-hydroxy-A -estratriene 1'7 carboxylate.

The aromatiza-tion of ring A by the use of ordinary Haney nickel takes :place substantially as follows. The selected 3(5) 5,19 trihydroxyetiocholanate is treated with ordinary Raney nickel under mild conditions and in the presence of a ketonic hydrogen acceptor. Any phenolic odor of the reaction product indicates that part of the ketonic hydrogen acceptor has become dehydrogenated to a phenol. The latter can be removed by extraction with a solution of 2N sodium hydroxide, and the residue subjected to a chromatographic purification.

When ethyl 3 (5) -5,l9,=trihydroxyetiocholanate was used as the starting material in the above process in the presence of cyclohexanone a hydrogen acceptor, three different compounds were isolated in a pure crystallineform.

The least polar substance, melting at 115-1 16", 7

chromatographic separation, was identified as ethyl 3-hydroxy-A -estratriene-17 carboxylate, melting at 176-178 (remelting at 184-185 It is to be noted that, though this substance is a phenol, it is not soluble in a solution of 2N sodium hydroxide. The ultraviolet absorption curve of this compound, lotted as molecular extinction coefficients, was practically identical with that of a reference sample of estradiol.

With tetranitromethane the substance gave an tainedfrom the early eluates was subjected to asolvolysis under mild conditions 'with'the purpo'se of achievinga deacetylation. Subsequent renewed chromatographic separation yielded'a very'smallamount of a crystalline fraction meltv ing at 217-219, whose ultraviolet absorption spectrum was determined in theregionbetween 200-and 300 millimicrons. There was-a maximum at 281 millimicrons and a'minimum' at 249 millimicrcns. This is characteristic for the series of natural estrogens in which ring A of the steroid nucleus'is aromatic. Hence it was suspected that the compound was methyl B-hydroxy-A estratriene-l7-carboxylate. The determination of a mixed melting point proved the identity of this compound with a sample of the same compound which has recently been preparedby Djerass'i and Scholz from methyl 3-keto-A etiocholadienate whichin turn has-been synthesized from cholesterol. I The invention may be more readily understoo by a consideration of the following illustrative examples wherein all temperatures areexpressed indegrees centigrade. It is to be understood,

.tions of water.

' sulfate the ether solution was brought to dryness, 30,

4 of course, the invention is not limited, but merely illustrated thereby.

Example ITreatment of ethyl 3(c),5,19-trihydroryetiocholanate with ordinary Raney nickel in the presence of cycZoheranona-To a total of 0.503 g. of pure ethyl 3(B),5,19-trihydroxyetiocholanate (melting between and 187), dissolved in 5 cc. of redistilled cyclohexanone (boiling point 148-150"), was added a suspension of approximately 2.12 g. of ordinary Raney nickel in 20.5 cc. of toluene. This mixture was stirred and refluxed continuously for a period of twentyfour hours, the bath temperature being kept at 128-132. Because part of the solvent had evaporated, 5 cc. of toluene each were added after eighteen and twenty-two hours respectively. After the termination of the reaction the solution was filtered from the Raney nickel and the latter washed with acetone. The filtrate was freed from solvents by first distilling it in an ordinary and finally in an oil pump vacuum (both at a temperature of 75). The oily residue had a strong phenolic odor. In order to remove any phenol present, the residue was dissolved in 200 cc. of redistilled ether and this solution washed successively'twice with 10 cc. portions of 2N sodium hydroxide and six times with 5 cc. por- After the drying with sodium eventually in an oil pump vacuum. The residue was a slightly yellow clear resin which resisted attempts at crystallization; weight 0.498 g.

The residue was dissolved in a mixture of 20 cc.'of benzene and 20 cc; of petroleum ether which was-chromatographed over a column (diameter-2.5 cm.) of 15 grams of alkali free'aluminum oxide (prepared by placing one part of aluminum oxide in an adsorption column, and then slowly washing with two parts of a 9:1 mix ture of methanol-glacial acetic acid, then washed acid free with methanol, and subsequently dried in an'oven at a temperature of 200 for a period of four hours). The absorbed material was eluted successively with 40 cc. portions of mixtures of benzene and petroleum ether (benzene content gradually increasing) benzene; mixtures of benzeneand ether (ether content gradually increasing) ether; mixtures of ether and chloroform (chloroform content gradually increasing) chloroform; mixtures of chloroform and methanol (methanol content gradually increasing); and finally methanol. A total of 0.495 g. of material was recovered. The major part of the material (0.430 g.) was contained in the 'eluates of benzene-petroleum ether, benzene, and benzene-ether. Though the residues were partly crystalline, apparently no efficient separation had been achieved. One of the chloroform-methanol (39:1) eluates furnished 7.1 mg. of a crystalline residue, which yielded needle-shaped crystals from acetone: melting point 209-211". They were not, further investigated. The test with tetranitromethane yielded no yellow color. This substance gave a; depression of the melting point when mixed withethyl 19-oxo-3 (p),,5 dihydroxyetiocholanate.

4 The major part of thematerial (0.430 g.) was subjected to a renewedchromatographic separa: tion,-for which it was dissolved in a mixture of 15cc. of benzene and 25- .c c.iof petroleum ether. The solution was filtered through a column (diameter 25 mm.) of 14 g. of alkali free aluminum oxide, prepared. as described above, within a period of thirty minutes. The eluateswere passed through, each within about fifteen minutes.

' Chromatographic 'fractzonat zon' N I Weightof 1 i 3 Solvent residue Appearance of residue i 29 .grams l v cc. benzene-{ cc. petr. ether 0.0047 Greasy.

(original solution) g 2 15 cc. benzene+25 cc. petr. ether 0.0361 Resmous. benzene+20 cc. petr. ether 0.0601 Crystalline.

benzene+20 cc. petr. ethers. 0. 0222 Do. 5 benzene+15 cc. petr. ether 0. 0207- Resinous.

. benzene+l5 cc. petr. ether.- 0. 0295 Pt. cryst;, pt. resinous. benzene+10 cc. petr. ether- 0.0361 Essentially crystalline. benzene-{-10 cc. petr. ether 0. 0204 Crystalline. benzene-F5 cc. petr. ether... 0. 0200 Essentially crystalline. benzene+5 cc. petr. ether- 0.0224 Few cryst. centers. h n n 0.0104 Resinous. benzene 0. 0113 Do. benzene+2 cc ether 0. 0166 Do benzene+3 cc ether- 0.0175 Pt. cryst., pt. resinous.

benezne+7 cc ethen. 0.0119 Do.' benzene+8 cc ethe 0.0106 Do. 31 cc benzene+9 cc ethe 0.0071 D0. cc. benzene+10 cc. ether. v 0. 0053. Resinous cc. each of benzene-ether mix- 0.0160 Do tures; ether content gradually e ns- 33 40 cc. ether 0.0023 Greasy. 34-38 40 cc. each of ether-methanol mix- 0.0165 Resinous.

f tures; methanol content gradually increasing. I 39 40 cc. methanol 0.0090 Crystalline.

Total 0. 4482 l The above chromatogram suggestedfthe pres ence of at least three different substances. H

Compound C22H32O4.-Fra.ctiOns 1 and -2 resisted attempts at crystallization. Fractions -3 and 4 were separately recrystallized bydissolving them in a small volume ofv ether towhichpetroleum ether was added. This caused the immedicrop; weight 0.9 mg.; melting point, 114.5-115.5.

Total pure material: 12.8 mg. The total yield can be somewhat increased by subjecting themother liquors, including the chromatographic fractions 2; and 5 to a renewed chromatographic fractionae tion. The substance (first crop) was transparent in the region between 200 and .300 millimicrons. The test with tetranitromethane yielded no yellowcolor. 1-

.Egrample 11- thyl .3 hydroxy A i -estrae triene -'17 carb mylate. The chromatographic fraction 5 resisted attempts at crystallization. Fractions 6'to ,9 were separately recrystallized by dissolving them in a small amount of ether and adding about twicethevolume of petroleum ether. This caused the separation of rosetteslof stout needles. Eventually 10 crystalline. fractions of identical material (determined by mixed melting points),...totalling 48.9 mg. with melting points between 175 and 178 (solidification and remelte Essentially crystalline Pt. cgyst. pt. resinous,

chloroform yielded with tetranitrom ethane an orange color. The ultraviolet absorption curve of this. compound plotted as molecular extinction coefficients, Was-practically identical with 2. referencesampleof estradiol. g a

The mother liquors resulting from the purification of the chromatographic fractions -6 tog 9 were combined with the whole fraction 10. On rechrornatographing this material only an additional 2.6 mg. of pure ethyl 3-hydroxy-AP3 estratriene-l7-carboxylate was obtained.

Example III. 3-hydromy-A -estratriene- 17-chrbowylic acid-6 milligrams of ethyl 3-hywithl cc. of water, 2 cc. of a solution of 'N so.-

ingvbetween 184 and 188) were obtained. Re-

dium carbonate and two 1 cc, portions of water. After drying with sodium sulfate, the ether phase yielded 0.9 mg. of neutral material. bonate phase, including the aqueous washings, was made acid to Congo paper by the addition of concentrated hydrochloric acid and the resulting precipitate brought into solution .by extracting four times with '5 cos. of ether. The combined ether phases were washed three times with 1 cc. of Water dried with sodium'sulfate and brought to dryness. Thus 3.5 mg. of crystalline acid material resulted which was recrystallized from small amounts of ether from which there occurred immediate crystallization of small cubes. First crop: 1.5 mg; melting point, 266-270 (turning dark brown). Second crop: 0.7 mg; melting point, 266-270? (turning dark brown). This substance did not give a depression of the melting point when it was mixed with an authentic sample ,of 3-hydroxy-A -estratriene-l7-carboxylic acid.

The chromatographic fractions 11 and 12 were The carnot investigated, and fraction 13 resisted attempts at crystallization.

Compound Cz2H34O4.F'ractions 14 to 20 yielded identical material which crystallized from ether in rosettes of long, stout, rectangular prisms. Eleven crystalline fractions, totalling 41.8 mg., had melting points between 134 and 137. 6 fractions, totalling 12.0 mg., yielded melting points between l28133. Renewed crystallization of either combined group raised the melting point to 136-137. The substance was transparent in the region between 200 and 300 millimicrons. The test with tetranitromethane gave no yellow color. [a] -+69.3 (7.4 mg, in 2.0 cc. of chloroform).

While the above examples illustrate the aromatization of ring A of the ethyl ester of 3(5),5,19-trihydroxyetiocholanic acid, it is to be understood any other ester form could be used in place of the ethyl ester, such as any other alkyl ester as the methyl, propyl, amyl, and the like ester; or an aryl ester, as the phenyl ester, and the like; or an aralkyl ester, as benzyl, and the like. Likewise, the 3(p),5,19-trihydroxy etiocholanate starting material may contain additional nuclear substituents so long as they do not interfere with the catalytic aromatization of ring A.

Also, the cyclohexanone used as a hydrogen acceptor may be replacedby other ket-onic hydrogenaccepto'rsprovided they are liquid compoundshavinga-relatively high boiling point. As examples ofother suitable ketone hydrogen "acceptors may be mentioned tetrachlorocyclohex anone, methyl cyclohexanone as'theZ-me'thyl "3-methyl-, or -4 methyl-oyclohexanone; ethyl cyclehexa'none'; 3;4,'5-trimethylor 3,5,5-trimethylcyclohexanone. 7 Other suitable hydrogen acceptors will readily suggest themselves to those skilled in this art. p

The following examples illustrate the aromatization of ring A by high-vacuum distillation.

Example IV.--M ethyl 3-hyd1oscy-A estratrz'ene-l7-carborylate.First experiment? A total of' 0.287 gram of dry 3(13),19-diacetoxy-5-hydroxyetiocholanic acid was heated inavacuum -(oil pump) to 170 and the temperature gradually raised to205". Durin this timethere was a persistent gas evolution in the retort. After this reaction had ceased, the reaction product was distilled in a high-vacuum (oil and mercury vapor pumps combined) at a temperature of 240- 265". :The distillate was a slightly greenish glass; weight: 0.234 gram; [a'] +3.3 (20.0 mg. in 2.0 cc; of chloroform). The residue in the retort was a light brown 'resin; weightz- 0.005' gram. Total loss of'weight: 0.048 gram- (15.7%); The distillate resisted attempts at recrystallization. 'A sample was subjected to a separation into acid and neutral 1 materialy only --sm-al l amounts of neutral material were obtained. The-distillate was transformed into the methyl ester by means oi 'diazomethane in an ethereal solution; 0.215 gram oi distillate yielded 2.225 gram of methyl ester. The latter :was a colorless glass which was dissolved in a mixture of 25 cc. of benzene and 7.5 cc. of petroleum ether and chromatographed over 10.0 g. of aluminum-oxide (diameter'of-col umn: 20 mm.). The adsorbate was'successively eluted with 25 cc. portions oibenzene-petroleum ether .(benzene. content gradually increasing); benzene; benzene-ether (ether content gradually increasing); ether; ether-methanol (methanol content gradually increasing). A substantial amount .(0.052 gram) was recovered from the benzene-petroleum ether fractions. One of them (0.023 gram) was crystalline. Recrystallization I from ether yielded several crystalline crops, totalling 12.4 mg. with melting points between 133 and 144?. The melting point of the purest fraction (4.9 mg.) was 143-144" and represented methyl 3(5),19-diacetoxy-A -etiocholanate. A solution in chloroform yielded a yellow color with tetranitromethane.

Second experiment: A total of 0.657 gram of 3(5) ,19-diacetoxy-5-hydroxyetiocholanic acid was quickly heated in a high-vacuum (oil and mercury vapor pumps combined) to a temperature of 153 which was-then slowly raised to 176. After the gas evolution had ceased, the material was distilled at a temperature of about 250. Total loss of weight; 0.124 grams (18.8%). The glassy distillate was recrystallized from ether. This furnished several crops of rosette arrangements of crystals totalling 0.138 gram; melting point between 1 44 and 150. The weight of the non-crystalline part was 0.372 gram. Recrystallization of .the combined crystalline fractions from ether furnished several crops of crys'talswith varying melting points; weight of the first crop: 0.041 gram; melting point, 158-161 The crystalline material was transformed into the methyl ester by means of diazomethane. The resulting resinous product was purified by chromatographic fractionation.-

From a total of 100 mg. of crude methyl ester, about-. 36 mg. could be recovered from the early eluates'lbenzene-petroleum other combinations). This material, which was resinous, was subjected to solvolysis with 0.5 equiv. potassium hydroxide in methanol at room temperature. This was done with the purpose of transforming any acetox'y groups lnto hydroxy groups. The resulting non-crystalline product was purified by chromatographic fractionation. As expected it had become more'polar. After first eluting with benzene-petroleum ether combinations and with benzene, approximately 11 mg. could be secured from the benzene-ether eluates. On recrystallizing from a mixture of acetone and petroleum ether, 1.6 mg. of feather-shaped crystals of methyl 3 -hydroxy-- {1135,10 estratriene 17 carboxylate, melting point 217-219", was obtained. A solution in chloroform gave, with tetranitromethane, a deep golden-yellow color. The ultraviolet absorption spectrum was determined in the region between 200 and 300 millimicrons. There was a maximum-at 281 'millimicrons and a minimum at 249 millimicrons. There was no depression of the melting point when mixed with an authentic sample of methyl 3-hydroxydl -estratriene 17-carboxylate.

Of course, many changes and variations can be made in the reaction conditions described above.- .For example, in place of the -3(,B).19- diacetox'yefiehydroxyetiocholanic acid starting material other cholanic acidderivatives may be .usedso long-as the 3- and 19-hydroxy groups are protected as byactylating with other suitable derivatives of aliphatic 'or aromatic carhoxylic acids.

9 and other reaction conditions may be varied within the limits obvious to those skilled in this art. 1

The aromatization of ring A of steroid compounds by either of the processes described above makes possible, for the first time, a means for obtainin from strophanthidin new compounds of the estrane type. Thus, a correlation between the cardiac aglycones and the hormones of the estrogen series has now been established.

It will be understood, with reference to the various compounds illustrated and described in this specification and its claims, that I do not intend that the invention of any or the compounds described or claimed shall be limited to any particular stereo-chemical configuration about any carbon atom and, in particular, about carbon atoms 3, 5, 10, 14 and 17. f

What I claim is:

1. A process for preparing compounds having the general formula wherein R is a lower alkyl radical, by aromatizing an ester of 3(p).5,19-trihydroxy etiocholanic REFERENCES CITED The following references are of record in the file of this patent:

Djerassi: Jour. Am. Chem. Soc. 69, 2404-2410 (1947). 

1. A PROCESS FOR PREPARING COMPOUNDS HAVING THE GENERAL FORMULA 